The Polymerase Chain Reaction (PCR) and real-time PCR have been widely accepted in the research and clinical fields as rapid and specific methods of detecting a target nucleic acid. However, the problem of non-specific amplification, i.e., amplification of non-target sequences, is often a limiting factor in achieving high sensitivity and specificity required for clinical applications. See Mackay, I. M. (2004) Real-time PCR in the microbiology laboratory, Clin. Microbiol. Infect. 10:190.
The non-specific amplification is thought to result from extension of primers annealed to secondary, partially complementary sites in the genome or to primer cross-annealing or self-annealing. The presence of non-specific extension products has been attributed to polymerase activity at ambient temperature where such partially complementary primer-template duplexes are stable. (Chou et al. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications Nucleic Acid Res. 20:1717). Accordingly, methods of inhibiting the primer extension activity of the polymerase at ambient temperature have been devised. These methods termed “hot start” assure that the polymerase becomes fully active only when the temperature is high enough to destabilize non-specific primer-template complexes so that extension of the primers at non-specific sites is avoided.
One hot start method involves an oligonucleotide that binds and inhibits the polymerase at low temperature, but not at high temperature. See Dang and Jayasena (1996) Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR J. Mol. Biol. 264:268. These oligonucleotides are extremely specific for and have high affinity to the target enzyme.
The hot-start approach however is not suitable for reverse transcription PCR (RT-PCR) applications where the reverse transcriptase requires temperatures below 50° C. Certain thermostable DNA polymerases have reverse transcription activity, allowing the use of a single enzyme to perform reverse transcription and amplification of cDNA (RT-PCR) in the same reaction mixture. See Myers and Gelfand (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase, Biochemistry 30:7661. However, RNA is labile at high temperature in the presence of divalent ions necessary for polymerase activity. For that reason, reverse transcription is carried out at lower temperature (50-60° C.) prior to commencement of the traditional PCR thermocycling. A typical oligonucleotide aptamer has melting temperature close to 60° C. and does not sufficiently release the inhibition of the polymerase at lower temperatures. It is therefore desirable to obtain a reversible polymerase inhibitor that could release inhibition at lower temperatures.